Regulation of transcription is largely focused on the initiation process that involves components of the basal transcription machinery (RNA polymerase II and the general transcription factors) and Mediator, a large (25 polypeptides, MW ~1 MDa) multi-subunit complex conserved throughout eukaryotes that is essential for transcriptional response to regulatory information. All of these components come together to form a giant preinitiation complex (over 60 different polypeptides, MW ~2.5 MDa) responsible for transcription initiation. Cryo-EM image analysis is uniquely suited to provide information about large and structurally dynamic complexes and our EM studies (along with the work of many others) have contributed significantly to the current understanding of transcription regulation. In this application we propose to apply advanced macromolecular cryo-electron microscopy (cryo-EM) and image analysis techniques to determine and analyze the structure of Mediator and investigate the mechanism of transcription initiation regulation. We propose to focus on obtaining structural information about the Head module, a structurally- and functionally-defined Mediator subcomplex that plays a critical role in interaction with RNA polymerase II (RNAPII) and in regulation of transcription initiation. Our specific aim is to undertake a detailed structural characterization of the Head module and its different conformational states, and to arrive at a description of the subunit organization of the module. The latter will be accomplished by taking advantage of a recent breakthrough in assembly of Head module and Head module subcomplexes from recombinant proteins. We will also identify factors that might influence Head module conformation and investigate the possible relevance of changes in Head module conformation for interaction with RNAPII Completion of these studies will provide structural information critical for unraveling the mechanism of transcription regulation.